1. Field of the Invention
Leptospirosis, caused by Leptospira interrogans, is a disease of animals and humans which has a worldwide distribution. L. interrogans is an immunologically diverse species and contains several distinct genetic groups. At least six serologically distinct types (serovars) have been identified in North America and about 190 serovars throughout the world. In North America, the most common cause of bovine leptospirosis is L. interrogans serovar hardjo-type hardjo-bovis. Hardjo-bovis and the reference strain for serovar hardjo, hardjoprajitno, are both associated in cattle with the causation of abortions, stillbirths, production of weak offspring, and infertility. In addition, cattle infected with serovar hardjo develop persistent renal infections and shed leptospires in their urine. Exposure to urine containing hardjo-bovis is considered to be the primary source of infections within herds.
The two hardjo types can be differentiated by restriction endonuclease analysis of genomic DNA. However, the existence of similar antigens shared by hardjo-bovis and hardjoprajitno prevents these two bacteria from being differentiated by classical serological techniques.
This invention relates to a sensitive diagnostic probe for distinguishing hardjo-bovis from other pathogenic leptospires, particularly those which commonly infect domestic animals in North America.
2. Description of the Prior Art
Diagnosis of leptospirosis usually depends upon demonstration of serum antibodies. The serologic method of choice is the microscopic agglutination test reported by Cole et al. [Appl. Microbiol. 25: 976-980 (1973)]. However, interpretation of microscopic agglutination test results is often subjective and is complicated by numerous factors, including previous vaccination or infection and antigenic heterogeneity among L. interrogans. Since cattle infected with hardjo-bovis may fail to produce detectable antibodies, an accurate diagnosis of infection with hardjo-bovis requires direct demonstration of L. interrogans in tissues, blood, or urine. This is achieved either by bacteriological culture or by immunological techniques. Isolation of serovar hardjo from clinical specimens is labor intensive and inconsistent and requires weeks or months before results are obtained. Similarly, antigens may be degraded or blocked in some clinical specimens and thus prevent immunological detection of bacteria.
Several investigators have utilized DNA-DNA hybridization methods for rapid and reliable detection of L. interrogans in biological samples (blood, urine, and tissue homogenates) [B. D. Millar et al., Vet. Microbiol. 15: 71-78 (1987); W. J. Terpstra et al. I, J. Gen. Microbiol. 133: 911-914 (1987); and W. J. Terpstra et al. II, J. Med. Microbiol. 22: 23-28 (1986)]. The probes for these hybridizations consist of genomic DNA labeled by nick translation with radiolabeled or biotinylated nucleotides. Although these probes are specific for L. interrogans, they demonstrate extensive cross-hybridization among pathogenic serovars (Terpstra et al. II, supra). LeFebvre [J. Clin. Microbiol. 25: 2236-2238 (1987)] and Van Eys et al. [J. Gen. Microbiol. 134: 567-574 (1988)] disclose cloned DNA probes which differentiate hardjo-bovis from hardjoprajitno in DNA blot hybridization studies. These probes have not been well characterized, nor used to detect hardjo-bovis in biological material. Whereas the probe described by Lefebvre recognizes a discrete fragment of the hardjo-bovis genome which apparently exists as a single copy, probes described by Van Eys et al. may exist as several copies in the hardjo-bovis genome. The restriction enzyme maps of the probes described by Van Eys et al. are distinct from the probes described herein and are likely to detect different sequences than the probes of the invention.